Lanthanide toolbox for organelle specific molecular imaging with multi-color

ABSTRACT

Provided herein are lanthanide complexes that exhibit specific subcellular localization to primary cilium. The lanthanide complexes provided herein are useful for imaging, tagging, and pull down of binding targets located in primary cilium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from and the benefit U.S. ProvisionalPatent Application Ser. No. 62/579,915 filed on Nov. 1, 2017, thedisclosure of which is hereby incorporated by reference in theirentirety.

FIELD OF INVENTION

The present disclosure relates to a cyclen-based lanthanide complexes,which exhibit specific subcellular localization in the primary cilium.The present disclosure has applications in organelle specific imagingand/or tagging/pull down.

BACKGROUND OF INVENTION

The primary cilium is a solitary, non-motile microtubule-based organellethat protrudes outwards from the surface of most normal eukaryoticcells. Upon in-depth investigation, it has been found to participateactively in various intercellular signaling pathways in mammals, e.g.Hedgehog (Hh), Wingless (Wnt) and PDGFRa, for cell migration,homeostasis, and cell cycle regulation. It also functions as themultisensory antenna of the cells towards external stimuli, such aschemical, temperature, and pressure stimuli.

Recently, a renaissance on the research of its structure was initiatedby substantial new and overwhelming evidence in support of itssignificant correlation with many human diseases and developmentaldisorders (collectively known as ciliopathies). For instance,dysfunctions of primary cilium signaling have been found to correlatestrongly in human polycystic kidney diseases, epithelial ovarian cancer,as well as aberrant skeletal development; the absence of primary ciliaand overexpression of proteins nearby have also well been observedthroughout the stages of pancreatic, breast, and prostate tumorigenesis.

That said, very little have the roles of primary cilium been clearly andfully known so far, with the lack of direct and specific imagingmethods, for example visible-to-near-infrared fluorescence imaging andmagnetic resonance imaging, being a critical factor. There are so manyorganelle-specific markers currently available for mitochondria, Golgiapparatus, and lysosome; however, visualization of primary cilia, todate, can only be achieved through immunostaining using antibodies orgreen fluorescent proteins, as no primary cilium-specific probes havebeen reported in literature. Such two indirect means are alwayschallenged with fixation and delivery issues, while auto-fluorescence isinevitable and the amount of information obtained through them islimited.

To address all the above problems, using a direct and specific imagingtool incorporated with lanthanide ions is a promising solution. The longemission lifetimes (micro to millisecond region), hypersensitive, sharpand fingerprint spectral profile of europium, paired with a time-gatedsystem, can effectively eliminate the interfering autofluorescence aswell as allowing the specific imaging of primary cattail in atime-resolved manner in vitro or in vivo.

Therefore, it turns out to be a need for simplicity of the design andsynthesis of a complex which exhibits the specific subcellularlocalization in the primary cilium and is optionally capable of proteintargeting, tagging and/or pull down.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art for the present application.

SUMMARY OF INVENTION

Accordingly, provided herein are cyclen-based lanthanide complexes,which exhibit specific subcellular localization in the primacy cilium.The landthanide complexes provided herein can be used for primary ciliumimaging as well as primary cilium tagging and/or pull down experiments.

In a first aspect, provided herein is a compound represented by formula(IV):

wherein m is an integer selected from 0, 1, 2 or 3;X is a pharmaceutically acceptable anion;Ln is Eu, Tb, Gd, Yb, Er, Dy, Sm, La, Ce, Pr, Nd, Pm, Tm or Y;A₁ is C, N, or Si;R₁, R₂ and R₃ are independently selected from the group consisting of—N(R₁₂)₂;

R₄, R₅, R₆, R₇, R₈, R₉ and R₁₀ are independently selected from the groupconsisting of H, alkyl, —CF₃, —OR₁₂, and —N(R₁₂)₂;R₁₁ is hydrogen, alkyl, aryl ether, —CO₂R₁₃; or —NH(C═O)R₁₄;R₁₂ and R₁₃ for each instance is independently selected from the groupconsisting of hydrogen and alkyl;R₁₄ is alkyl, cycloalkyl, aryl, or heteroaryl; andn is a whole number selected from 1-8, wherein one of R₁, R₂ and R₃ isnot N(R₁₂)₂.

In a first embodiment of the first aspect, provided herein is thecompound of the first aspect, wherein R₁ and R₃ are independently—N(H)(alkyl).

In a second embodiment of the first aspect, provided herein is thecompound of the first embodiment of the first aspect, wherein A₁ is C.

In a third embodiment of the first aspect, provided herein is thecompound of the first embodiment of the first aspect, wherein n is 4, 5,or 6.

In a fourth embodiment of the first aspect, provided herein is thecompound of the third embodiment of the first aspect wherein R₁₁ is—NH(C═O)Ar.

In a fifth embodiment of the first aspect, provided herein is thecompound of the first aspect, wherein the compound is represented by theformula (V):

wherein m is an integer selected from 0, 1, 2 or 3;X is a pharmaceutically acceptable anion;Ln is Eu, Tb, Gd, Yb, Er, Dy, Sm, La, Ce, Pr, Nd, Pm, Tm or Y;R₂ is selected from the group consisting of:

R₁₂ for each instance is independently alkyl; andn is a whole number selected from 1-8.

In a sixth embodiment of the first aspect, provided herein is thecompound of the fifth embodiment of the first aspect, wherein eachinstance of R₁₂ is tert-butyl.

In a seventh embodiment of the first aspect, provided herein is thecompound of the sixth embodiment of the first aspect, wherein n is 4, 5,or 6.

In an eighth embodiment of the first aspect, provided herein is thecompound of the first aspect, wherein the compound is selected from thegroup consisting of:

wherein Ln is Eu, Tb, Gd, Yb, Er, Dy, Sm, La, Ce, Pr, Nd, Pm, Tm or Y;andX is a pharmaceutically acceptable anion.

In a ninth embodiment of the first aspect, provided herein is thecompound of the first aspect, wherein the molecule selectively binds toprimary cilium.

In a second aspect, provided herein is a method for imaging primarycilium in a biological cell comprising the steps of contacting thebiological cell with the compound of the first aspect and imaging thebiological cell.

In a first embodiment of the first aspect, provided herein is the methodof the first aspect, wherein the imaging is performed using linearfluorescence microscopy using UV light excitation or a two-photonconfocal laser scanning microscope.

In a second embodiment of the first aspect, provided herein is themethod of the first aspect, wherein the imaging is done in livingbiological cells.

In a third aspect, provided herein is a method for preparing a compoundof the eighth embodiment of the first aspect, comprising the step of

a) contacting a compound of formula (VI):

with a compound selected from the group consisting of:

thereby forming the compound of the eighth embodiment of the firstaspect.

Throughout this specification, unless the context requires otherwise,the word “include” or “comprise” or variations such as “includes” or“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. It is also noted that in thisdisclosure and particularly in the claims and/or paragraphs, terms suchas “included”, “comprises”, “comprised”, “comprising” and the like canhave the meaning attributed to it in U.S. Patent law; e.g., they canmean “includes”, “included”, “including”, and the like; and that termssuch as “consisting essentially of” and “consists essentially of” havethe meaning ascribed to them in U.S. Patent law, e.g., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of thepresent invention.

Furthermore, throughout the specification and claims, unless the contextrequires otherwise, the word “include” or variations such as “includes”or “including”, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups, in certain embodiments,a straight chain or branched chain alkyl has about 30 or fewer carbonatoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ forbranched chain), and alternatively, about 20 or fewer. Likewise,cycloalkyls have from about 3 to about 10 carbon atoms in their ringstructure, and alternatively about 5, 6 or 7 carbons in the ringstructure.

The term “aryl” is art-recognized and refers to 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, naphthalene, anthracene, pyrene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics.” The aromaticring may be substituted at one or more ring positions with suchsubstituents as described above, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, or thelike. The term “aryl” also includes polycyclic ring systems having twoor more cyclic rings in which two or more carbons are common to twoadjoining rings (the rings are “fused rings”) wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings may be cycloalkyls,cycloalkenyls, cycloalknyls, aryls and/or heterocyclyls.

Other definitions for selected terms used herein may be found within thedetailed description of the present invention and apply throughout.Unless otherwise defined, all other technical terms used herein have thesame meaning as commonly understood to one of ordinary skill in the artto which the present invention belongs.

Other aspects and advantages of the present invention will be apparentto those skilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The above and other objects and features of the present invention willbecome apparent from the following description of the present invention,when taken in conjunction with the accompanying drawings, in which:

FIG. 1A shows the chemical structures and emission spectra of HGEu001 inaqueous solution (10 μM and λ_(ex)=340 nm).

FIG. 1B shows the chemical structures and emission spectra of HGEu002 inaqueous solution (10 μM and λ_(ex)=340 nm).

FIG. 2A shows Linear fluorescence microscopy images of the red emissionfrom HGEu001 (dosage concentration=10 μM, λ_(ex)=375 nm, bandpass=610-630 nm) after 6 hours exposure in HeLa cells.

FIG. 2B shows Linear fluorescence microscopy images of the red emissionfrom HGEu001 (dosage concentration=10 μM, λ_(ex)=375 nm, bandpass=610-630 nm) after 6 hours exposure in HeLa cells.

FIG. 2C shows Linear fluorescence microscopy images of the red emissionfrom HGEu001 (dosage concentration=10 μM, λ_(ex)=375 nm, bandpass=610-630 nm) after 6 hours exposure in HeLa cells.

FIG. 2D shows Linear fluorescence microscopy images of the GolgiTracker®Oregon Green (W6748, Wheat Germ Agglutinin) dosed in HeLa cells (50 nM,λ_(ex)=488 nm, band pass=505-555 nm.

FIG. 2E shows Linear fluorescence microscopy images of the LysoTracker®Green DND-26 (L-7526) dosed in HeLa cells (50 nM, λ_(ex)=488 nm, bandpass=505-555 nm).

FIG. 2F shows Linear fluorescence microscopy images of the MitoTracker®Green FM (M-7514) dosed in HeLa cells (50 nM, λ_(ex)=488 nm, bandpass=505-555 nm).

FIG. 2G shows Linear fluorescence microscopy merged images for FIGS. 2Aand 2D.

FIG. 2H shows Linear fluorescence microscopy merged images for FIGS. 2Band 2E.

FIG. 2I shows Linear fluorescence microscopy merged images for FIGS. 2Cand 2F.

FIG. 3A shows the two-photon time lapses in vitro images of HGEu001 inHeLa cells of which the images were taken at 3 hours incubation timepoints. The red emission of HGEu001 (arrows) is localized in primarycilium.

FIG. 3B shows the two-photon time lapses in vitro images of HGEu001 inHeLa cells of which the images were taken at 6 hours incubation timepoints. The red emission of HGEu001 (arrows) is localized in primarycilium.

FIG. 3C shows the two-photon time lapses in vitro images of HGEu001 inHeLa cells of which the images were taken at 18 hours incubation timepoints. The red emission of HGEu001 (arrows) is localized in primarycilium.

FIG. 3D shows the two-photon time lapses in vitro images of HGEu001 inHeLa cells of which the images were taken at 24 hours incubation timepoints. The red emission of HGEu001 (arrows) is localized in primarycilium.

FIG. 3E shows the overlay images of fluorescence channel shown in FIG.3A and bright field channel (Dosed concentration=10 μM, λ_(ex)=700 nm,filter Bandpass=550-665 nm). The red emission of HGEu001 (arrows) islocalized in primary cilium.

FIG. 3F shows the overlay images of fluorescence channel shown in FIG.3B and bright field channel (Dosed concentration=10 μM, λ_(ex)=700 nm,filter Bandpass=550-665 nm). The red emission of HGEu001 (arrows) islocalized in primary cilium.

FIG. 3G shows the overlay images of fluorescence channel shown in FIG.3C and bright field channel (Dosed concentration=10 μM, λ_(ex)=700 nm,filter Bandpass=550-665 nm). The red emission of HGEu001 (arrows) islocalized in primary cilium.

FIG. 3H shows the overlay images of fluorescence channel shown in FIG.3D and bright field channel (Dosed concentration=10 μM, λ_(ex)=700 nm,filter Bandpass=550-665 nm). The red emission of HGEu001 (arrows) islocalized in primary cilium.

FIG. 4A shows the co-staining experiments of red HGEu001 in fluorescencemicroscope with marked distance for primary cilium (Green line) (Dosedconcentration 10 μM HGEu001, 6 hours incubation after GFP-ARL13B wastransfected and expressed).

FIG. 4B shows the green GFP-ARL13B in fluorescence microscope withmarked distance for primary cilium (Green line).

FIG. 4C shows the overlay of FIGS. 4A and 4B in fluorescence microscopewith marked distance for primary cilium (Green line).

FIG. 4D shows the intensity of HGEu001.

FIG. 4E shows the co-staining experiments of red HGEu002 (control) influorescence microscope with marked distance for primary cilium (Greenline) (Dosed concentration 10 μM HGEu002, 6 hours incubation afterGFP-ARL13B was transfected and expressed).

FIG. 4F shows the green GFP-ARL13B in fluorescence microscope withmarked distance for primary cilium (Green line).

FIG. 4G shows the overlay of FIGS. 4E and 4F in fluorescence microscopewith marked distance for primary cilium (Green line).

FIG. 4H shows the intensity of HGEu002.

FIG. 5A shows the three dimensional in vitro imaging and emissionspectra of 10 μM in xz plane for HGEu001 incubated in HeLa cells for 6hours. (λ_(ex)700 nm).

FIG. 5B shows the three dimensional in vitro imaging and emissionspectra of 10 μM in xz plane for HGEu002 incubated in HeLa cells for 6hours. (λ_(ex)700 nm).

FIG. 5C shows the three dimensional in vitro imaging and emissionspectra of 10 μM in xy plane for HGEu001 incubated in HeLa cells for 6hours. (λ_(ex)700 nm).

FIG. 5D shows the three dimensional in vitro imaging and emissionspectra of 10 μM in xy plane for HGEu002 incubated in HeLa cells for 6hours. (λ_(ex)700 nm).

FIG. 5E shows the three dimensional in vitro imaging and emissionspectra of 10 μM in xyz plane for HGEu001 incubated in HeLa cells for 6hours. (λ_(ex)700 nm).

FIG. 5F shows the three dimensional in vitro imaging and emissionspectra of 10 μM in xyz plane for HGEu002 incubated in HeLa cells for 6hours. (λ_(ex)700 nm).

FIG. 6A shows the three dimensional (by z stack) two-photon confocal invitro images of HGEu001 with co-localization of greenGFP-ARL14B/GFP-IFT88/MitoTracker® Green FM (M-7514) in HeLa cells.(λ_(ex)=700 nm) HeLa cells were first transfected withGFP-ARL13B/GFP-IFT88 or incubated with MitoTracker® Green FM (M-7514)for 15 minutes and further incubated 6 hours with 10 μM of HGEu001.

FIG. 6B shows the three dimensional (by z stack) two-photon confocal invitro images of HGEu002 (negative control) with co-localization of greenGFP-ARL14B/GFP-IFT88/MitoTracker® Green FM (M-7514) in HeLa cells.(λ_(ex)=700 nm) HeLa cells were first transfected withGFP-ARL13B/GFP-IFT88 or incubated with MitoTracker® Green FM (M-7514)for 15 minutes and further incubated 6 hours with 10 μM of HGEu002.

FIG. 6C shows the three dimensional (by z stack) two-photon confocal invitro images of Dark field of HGEu001 with co-localization of greenGFP-ARL14B/GFP-IFT88/MitoTracker® Green FM (M-7514) in HeLa cells.(λ_(ex)=700 nm) HeLa cells were first transfected withGFP-ARL13B/GFP-IFT88 or incubated with MitoTracker® Green FM (M-7514)for 15 minutes and further incubated 6 hours with 10 μM of HGEu001.

FIG. 6D shows the three dimensional (by z stack) two-photon confocalvitro images of Dark field of HGEu002 (negative control) withco-localization of green GFP-ARL14B/GFP-IFT88/MitoTracker® Green FM(M-7514) in HeLa cells. (λ_(ex)=700 nm) HeLa cells were firsttransfected with GFP-ARL13B/GFP-IFT88 or incubated with MitoTracker®Green FM (M-7514) for 15 minutes and further incubated 6 hours with 10μM of HGEu002.

FIG. 7A shows HPLC spectra of HGEu001.

FIG. 7B shows HPLC spectra of HGEu002.

FIG. 8 shows HRMS(+ESI) spectrum of the complex HGEu001. (m/z calcd. forC47H65EuN9O4 [M−H2O−2H]+972.4372, found 972.4378, calcd. forC47H66ClEuN9O4 [M−H2O−H+Cl—]+1008.4139, found 1008.4119, calcd. forC47H67Cl2EuN9O4 [M−H2O+2O+2Cl]+1044.3905, found 1044.3882).

FIG. 9 shows HRMS(+ESI) spectrum of the complex HGEu002. (m/z calcd. forC48H65EuF3N9O4 [M−H2O−H]²⁺ m/z=1041.4324/2=520.7162, found 520.7186.

FIG. 10 shows the absorption spectra of HGEu001 and HGEu002 in aqueoussolution (10 μM).

FIG. 11A shows the emission spectra of HGEu001 aqueous solution (10 μM,λ_(ex)=340 nm). The spectra were recorded on Horiba Flurolog-3spectrophotometer. The same emission bands and ratios obtained arecompared with the emission spectra measured with Edinburgh instrumentFLS920 spectrophotometer as in FIGS. 1A-1B.

FIG. 11B shows the emission spectra of HGEu002 aqueous solution (10 μM,λ_(ex)=340 nm). The spectra were recorded on Horiba Flurolog-3spectrophotometer. The same emission bands and ratios obtained arecompared with the emission spectra measured with Edinburgh instrumentFLS920 spectrophotometer as in FIGS. 1A-1B.

FIG. 12A shows the emission decay curve of the complex HGEu001 and inD₂O and H₂O. (λ_(em)=614 nm. ⁵D₀→⁷F₂. λ_(ex)=355 nm).

FIG. 12B shows the emission decay curve of the complex HGEu002 in D₂Oand H₂O. (λ_(em)=614 nm. ⁵D₀→⁷F₂. λ_(ex)=355 nm).

FIG. 13A shows the raw data of cytotoxicity of HGEu001 in Table 3.

FIG. 13B shows the raw data of cytotoxicity of HGEu002 in Table 3.

FIG. 14A shows the two-photon time lapses in vitro images of HGEu002 inHeLa cells which the images were taken at 3 hours incubation timepoints; The red emission of HGEu002 is localized in cytoplasm. (Inparallel with FIG. 3A).

FIG. 14B shows the two-photon time lapses in vitro images of HGEu002 inHeLa cells which the images were taken at 6 hours incubation timepoints; The red emission of HGEu002 is localized in cytoplasm. (Inparallel with FIG. 3B).

FIG. 14C shows the two-photon time lapses in vitro images of HGEu002 inHeLa cells which the images were taken at 18 hours incubation timepoints; The red emission of HGEu002 is localized in cytoplasm. (Inparallel with FIG. 3C).

FIG. 14D shows the two-photon time lapses in vitro images of HGEu002 inHeLa cells which the images were taken at 24 hours incubation timepoints; The red emission of HGEu002 is localized in cytoplasm. (Inparallel with FIG. 3D).

FIG. 14E shows the overlay images of fluorescence channel shown in FIG.14A and bright field channel (Dosed concentration=10 μM, λ_(ex)=700 nm,filter Bandpass=550-665 nm); The red emission of HGEu002 is localized incytoplasm. (In parallel with FIG. 3E).

FIG. 14F shows the overlay images of fluorescence channel shown in FIG.14B and bright field channel (Dosed concentration=10 μM, λ_(ex)=700 nm,filter Bandpass=550-665 nm); The red emission of HGEu002 is localized incytoplasm. (In parallel with FIG. 3F).

FIG. 14G shows the overlay images of fluorescence channel shown in FIG.14C and bright field channel (Dosed concentration=10 μM, λ_(ex)=700 nm,filter Bandpass=550-665 nm); The red emission of HGEu002 is localized incytoplasm. (In parallel with FIG. 3G).

FIG. 14H shows the overlay images of fluorescence channel shown in FIG.14D and bright field channel (Dosed concentration=10 μM, λ_(ex)=700 nm,filter Bandpass=550-665 nm); The red emission of HGEu002 is localized incytoplasm. (In parallel with FIG. 3H).

FIG. 15A shows the three dimensional in vitro imaging of 10 μM in xzplan for HGEu001 incubated in MRC-5 cells with 6 hours. (λ_(ex)=700 nm,In parallel with FIG. 5A).

FIG. 15B shows the three dimensional in vitro imaging of 10 μM in xzplan for HGEu002 incubated in MRC-5 cells with 6 hours. (λ_(ex)=700 nm,In parallel with FIG. 5B).

FIG. 15C shows the three dimensional in vitro imaging of 10 μM in xyplan for HGEu001 incubated in MRC-5 cells with 6 hours. (λ_(ex)=700 nm,In parallel with FIG. 5C).

FIG. 15D shows the three dimensional in vitro imaging of 10 μM in xyplan for HGEu002 incubated in MRC-5 cells with 6 hours. (λ_(ex)=700 nm,in parallel with FIG. 5D).

FIG. 15E shows the three dimensional in vitro imaging of 10 μM in xyzplane for HGEu001 incubated in MRC-5 cells with 6 hours. (λ_(ex)=700 nm,In parallel with FIG. 5E).

FIG. 15F shows the three dimensional in vitro imaging of 10 μM in xyzplane for HGEu002 incubated in MRC-5 cells with 6 hours. (λ_(ex)=700 nm,In parallel with FIG. 5F).

FIG. 16 shows H NMR spectrum of compound 2. (400 MHz, DMSO-d₆).

FIG. 17 shows C NMR spectrum of compound 2. (100 MHz, DMSO-d₆).

FIG. 18 shows H NMR spectrum of compound 3. (400 MHz, DMSO-d₆).

FIG. 19 shows C NMR spectrum of compound 3. (100 MHz, DMSO-d₆)

FIG. 20 shows H NMR spectrum of compound 4. (400 MHz, DMSO-d₆)

FIG. 21 shows C NMR spectrum of compound 4. (100 MHz, DMSO-d₆)

FIG. 22 shows H NMR spectrum of compound 5. (400 MHz, CDCl₃)

FIG. 23 shows C NMR spectrum of compound 5. (100 MHz, CDCl₃)

FIG. 24 shows H NMR spectrum of HGL001. (400 MHz, DMSO-d₆)

FIG. 25 shows C NMR spectrum of HGL001. (100 MHz, DMSO-d₆)

FIG. 26 shows H NMR spectrum of HGL002. (400 MHz, DMSO-d₆)

FIG. 27 shows C NMR spectrum of HGL002. (100 MHz, DMSO-d₆)

FIG. 28 shows molecular library of the europium complexes which havesimilar structures with HGEu001 for primary cilium specific imagingscreening.

FIG. 29 shows the synthesis scheme (Scheme 1) of HGEu001 and its motifcomplex HGEu002: (a) (4-ethynylpyridin-2-yl)methanol, Pd(PPh₃)₄, CuI,DIPEA THF; (b) MsCl, DIPEA, DCM; (c) cyclen, NaHCO₃, CH₃CN; (d) K₂CO₃,MeCN, 60° C.; (e) EuCl₃.6H₂O, MeOH, rt., 24 hours.

FIG. 30 shows the chemical structures of the new lanthanide complexesfor organelle specific imaging.

FIG. 31a shows the absorption spectrum of HGEu001-Coumarin in aqueoussolution (10 μM).

FIG. 31b shows the emission spectrum of HGEu001-Coumarin underexcitation of 330 in aqueous solution (10 μM).

FIG. 31c shows the emission spectrum of HGEu001-Coumarin underexcitation of 430 in aqueous solution (10 μM).

FIG. 32a shows the absorption spectrum of HGEu001-Por in aqueoussolution (10 μM).

FIG. 32b shows the emission spectrum of HGEu001-Por under excitation of330 in aqueous solution (10 μM).

FIG. 32c shows the emission spectrum of HGEu001-Por under excitation of430 in aqueous solution (10 μM).

FIG. 33 shows the chemical structures of HGEu001-Biotin andHGEu001-EDECRIN for primary cilium related protein enrichment andlabeling respectively.

FIG. 34 shows the SDS gel imaging of HGEu0001-Biotin and HGEu001-EDECRINof cell lysate.

FIG. 35 shows the protein number that identified by proteomic analysisof proteins in SDS gels band. 44 primary cilium related proteins areidentified by the two probes HGEu001-Biotin and HGEu001-EDECRIN.

FIG. 36 shows the mass spectrum of the observed peptide sequence‘NSIMKCDVDIRKDLY ANTVLSGGTTMYPGIADR’ which labeled with HGEu001-EDECRINfrom proteomic analysis.

FIG. 37a depicts an exemplary synthetic scheme for the preparation ofthe intermediate HGEu001-NH₂ that can be used in the preparation ofHGEu001-Biotin, HGEu001-EDECRIN, HGEu001-Por, and HGEu001-Coumarin,wherein the synthetic steps call for: a) MsCl, DIPEA, DCM; b) NaHCO₃,ACN; c) K₂CO₃, ACN; d) 6 M HO; e) EuCl₃.6H₂O.

FIG. 37b depicts an exemplary synthetic scheme for the preparationHGEu001-Biotin, HGEu001-EDECRIN, HGEu001-Por, and HGEu001-Coumarin fromintermediate HGEu001-NH₂, wherein the synthetic steps call for: f)Coumarin-NHS, DIPEA, DMSO; g) Por-NHS, DIPEA, DMSO; h) EDECRIN-NHS,DIPEA, DMSO; i) Biotin-NHS, DIPEA, DMSO.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compounds of formula (IV) and formula (V), whichexhibit specific subcellular localization in the primary cilium withblue to deep red emission color under ultraviolet (UV)/visible/nearinfrared (NM) light excitation. The emission properties are tunable bythe selection of the function group attached in the parent primarycilium marker (tris(N-(tert-butyl)acetamide) cy den-based lanthanidecomplexes (molecules of formula (I), (II), and (III))). The presentdisclosure has applications in organelle specific imaging and proteinquantitative analysis. The compounds of formula (IV) and (V) are analogsof tris(N-(tert-butyl)acetamide) cyclen-based lanthanide complexes(molecules of formula (I), (II), and (III) described U.S. patentapplication Ser. No. 15/604,660, which is hereby incorporated in itsentirety) and extend the utility of the molecules of formula (I), (II),and (III) and surprisingly maintain many of their advantageousproperties, such as cilium binding specificity, specific subcellularlocalization, high water solubility, and high stability.

Molecules of formula (I) have been shown to be useful for imagingrod-like organelle in biological cells, wherein the molecule of formula(I) comprises:

wherein Ln is selected from Eu, Tb, Gd, Yb, Er, Dy, Sm, La, Ce, Pr, Nd,Pm, Tm and Y, X⁻ is selected from Cl⁻, NO₃ ⁻, CH₃COO⁻, ClO₄ ⁻ or otheranions; A₁ is selected from C, N or Si; R₁, R₂ and R₃ are jointly orseparately selected from NH(tert)Bu, OH⁻ or other amine; R₄, R₅, R₆, R₇,R₈, R₉ and R₁₀ are jointly or separately selected from H, CF₃, OMe, OEt,OH or NMe₂; R₁₁ is selected from alkyl, aryl ether, ester, amide oraromatic rings; m is an integer selected from 0, 1, 2 or 3. Inparticular embodiments, the derivatives of the molecule of the formula(I) can be represented by formula (II):

wherein R₁₁ is selected from:

andwherein R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₁₈, R₁₉, and R₂₀ are jointly orseparately selected from H, CF₃, OMe, OEt, OH or NMe₂; A₂ is selectedfrom C, N, or Si. Alternatively, the corresponding substituents for X,A₂ and R_(n) (where n=4-20) in each of the derivatives can be defined asfollows:X=Cl, A₂=C, R_(n)=H (n=4-10, 12-16);  HGL001X=Cl, A₂=C, R_(n)=H (n=4-10, 12-15), R₁₆=CF₃;  HGL002X=Cl, A₂=C, R_(n)=H (n=4-10, 12-15), R₁₆=OMe;  HGL003X=Cl, A₂=C, R_(n)=H (n=4-10, 12-15), R₁₀=NMe₂;  HGL004X=Cl, A₂=N, R_(n)=H (n=4-10, 12-15);  HGL005X=Cl, R_(n)=H (n=4-10, 17-19);  HGL006X=Cl, R_(n)=H (n=4-10), R₂₀=OH;  HGL007X=Cl, R_(n)=H (n=4-10), R₂₀=OEt;  HGL008X=Cl, A₂=C, R_(n)=H (n=4-6, 8, 10, 12-16), R₇=R₉=OMe;  HGL009X=Cl, A₂=C, R_(n)=H (n=4-6, 8, 10, 12-15), R₇=R₉=OMe, R₁₆=CF₃;  HGL010X=Cl, A₂=C, R_(n)=H (n=4-6, 8, 10, 12-15), R₇=R₉=R₁₆=OMe;  HGL011X=Cl, A₂=C, R_(n)=H (n=4-6, 8, 10, 12-15), R₇=R₉=OME, R₁₆=NMe₂;  HGL012X=Cl, A₂=N, R_(n)=H (n=4-6, 8, 10, 12-15), R₇=R₉=OMe;  HGL013X=Cl, A₂=C, R_(n)=H (n=4-6, 8, 10, 17-19), R₇=R₉=OMe;  HGL014X=Cl, A₂=C, R_(n)=H (n=4-6, 8, 10), R₇=R₉=OMe, R₂₀=OH;  HGL015X=Cl, A₂=C, R_(n)=H (n=4-6, 8, 10), R₇=R₉=OMe, R₂₀=OEt;  HGL016where Ln refers to lanthanide; OMe refers to a methoxy group; NMe₂refers to a nitro-dimethyl group; OEt refers to ethoxy group.

Additional analogs of the molecules (I), (II), and (III) have beendeveloped that further extend the utility of these molecules asdescribed herein. It has been surprisingly discovered that the moleculesof (I), (II), and (III) can be modified to include reactivefunctionality capable of protein targeting/pull down or additionalfluorescent groups expanding the excitation and emission wavelengths ofthe molecules, without impacting their ability to localize andselectively bind to cilium present in cells.

In a first aspect, provided herein is a compound represented by theformula (IV):

wherein in is an integer selected from 0, 1, 2 or 3;X is a pharmaceutically acceptable anion;Ln is Eu, Tb, Gd, Yb, Er, Dy, Sm, La, Ce, Pr, Nd, Pm, Tm or Y;A₁ is C, N, or Si;R₁, R₂ and R₃ are independently selected from the group consisting of—N(R₁₂)₂;

R₄, R₅, R₆, R₇, R₈, R₉ and R₁₀ are independently selected from the groupconsisting of H, alkyl, —CF₃, —OR₁₂, and —N(R₁₂)₂;R₁₁ is hydrogen, alkyl, aryl ether, —CO₂R₁₃; or —NH(C═O)R₁₄;R₁₂ and R₁₃ for each instance is independently selected from the groupconsisting of hydrogen and alkyl;R₁₄ is alkyl, cycloalkyl, aryl, or heteroalyl; andn is a whole number selected from 1-8, wherein one of R₁, R₂ and R₃ isnot N(R₁₂)₂.

In instances in which one of group member of R₁, R₂ and R₃ is notN(R₁₂)₂, the one group member of R₁, R₂ and R₃ that is not is N(R₁₂)₂ isselected from the group consisting of:

and the remaining two group members of R₁, R₂ and R₃ are eachindependently —N(R₁₂)₂.

In certain embodiments, R₁ and R₃ are independently —N(H)(alkyl); R₁ andR₂ are independently —N(H)(alkyl); or R₂ and R₃ are independently—N(H)(alkyl).

In certain embodiments, R₁ and R₃ are independently —N(H)(alkyl) and R₂is selected from the group consisting of:

In certain embodiments, the compound of formula (IV) can be representedby the compound of formula (V):

wherein m is an integer selected from 0, 1, 2 or 3;X is a pharmaceutically acceptable anion;Ln is Eu, Tb, Gd, Yb, Er, Dy, Sm, La, Ce, Pr, Nd, Pm, Tm or Y;R₂ is selected from the group consisting of:

R₁₂ for each instance is independently alkyl; andn is a whole number selected from 1-8.

As used herein, the term “pharmaceutically acceptable anion” refers tothe relatively non-toxic, organic or inorganic anions including, but arenot limited to, chloride, bromide, iodide, perchlorate, carbonate,bicarbonate, sulfate, phosphate, monohydrogen phosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, acetate, maleate, fumarate,formate, malonate, oxalate, lactate, tartrate, citrate, gluconate,mesylate, besylate, tosylate, succinate, and salicylate sulfate,sulfite, bisulfate, bisulfite, nitrate and nitrite. In certainembodiments, X is selected from the group consisting of chloride,nitrate, acetate, and perchlorate.

In certain embodiments, Ln is Eu, Tb, Gd, Yb, Er, Dy, Sm, La, Ce, Pr,Nd, Pm, Tm or Y, wherein Ln is in the +2, +3, or +4 oxidation state. Incertain embodiments. Ln is selected from the group consisting ofEu(III), Tb(III), Gd(III), Yb(III), Er(III), Dy(III), Sm(III), La(III),Ce(III), Pr(III), Nd(III), Pm(III), Tm(III) and Y(III). In certainembodiments, Ln is Eu(III).

In certain embodiments, A₁ is C or N. In certain embodiments, A₁ is C.

In certain embodiments, n is a whole number selected from the groupconsisting of 2-8; 3-8; 4-8; 4-6; 5-8; 5-7; or 6-8. In certainembodiments, n is 6.

In certain embodiments, R₁₁ is —NH(C═O)Ar. In such embodiments, Ar canbe represented by

wherein R₁₂, R₁₃, R₁₄, R₁₅, and R₁₆, are independently selected from H,CF₃, OMe, OEt, OH or NMe₂; or wherein A₂ is N, R₁₆ is absent; and A₂ isselected from C, N, or Si. In certain embodiments, R₁₂, R₁₃, R₁₄, andR₁₅ are H; and R₁₆ is CF₃, OMe, or NMe₂.In certain embodiments, R₁₂ is independently for each instance a C₁-C₆alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2-methylpentane,3-methylpentane, 2,3-dimethylbutane, 2,2-dimethylbutane, and the like.

In certain embodiments, the compound of formula (IV) is selected fromthe group consisting of:

wherein Ln is Eu, Tb, Gd, Yb, Er, Dy, Sm, La, Ce, Pr, Nd, Pm, Tm or Y,wherein Ln is in the +2, +3, or +4 oxidation state; and X is chloride,bromide, iodide, perchlorate, carbonate, bicarbonate, sulfate,phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate,pyrophosphate, acetate, maleate, fumarase, formate, malonate, oxalate,lactate, tartrate, citrate, gluconate, mesylate, besylate, tosylate,succinate, and salicylate sulfate, sulfite, bisulfate, bisulfite,nitrate or nitrite. In certain embodiments, X is selected from the groupconsisting of chloride, nitrate, acetate, and perchlorate.

In certain embodiments, the compound of formula (IV) is selected fromthe group consisting of:

wherein Ln is Eu; and X⁻ is Cl⁻.

In certain embodiments, there is provided a compound for imagingrod-like organelle in biological cells comprising any of the compoundsdescribed herein, wherein the rod-like organelle in biological cells isprimary cilium.

In certain embodiments, there is provided a compound for imagingrod-like organelle in biological cells comprising any of the compoundsdescribed herein, wherein the imaging is done in living cells.

In certain embodiments, there is provided a compound for imagingrod-like organelle in biological cells comprising any of the compoundsdescribed herein, wherein the imaging is performed using a linearfluorescence microscopy under UV light excitation or a two-photonconfocal laser scanning microscope.

In certain embodiments, there is provided a compound for imagingrod-like organelle in biological cells comprising a compound selectedfrom the group consisting of:

wherein Ln is Eu(III); and X is selected from the group consisting ofchloride, nitrate, acetate, and perchlorate.

In certain embodiments, there is provided a compound for imagingrod-like organelle in biological cells comprising a compound describedherein, wherein the compound can selectively bind to proteins in cells.In certain embodiments, the protein is at least one primary ciliumrelated protein.

In certain embodiments, there is provided a compound for imagingrod-like organelle in biological cells comprising a compound describedherein being used for quantitative analysis of primary cilium relatedproteins.

In certain embodiments, there is provided a compound for imagingrod-like organelle in biological cells comprising a compound describedherein being used as a disease diagnosis probes.

In certain embodiments, there is provided a compound for imagingrod-like organelle in biological cells comprising a compound describedherein being used as an organelle targeting specific vector.

In certain embodiments, there is provided a compound for imagingrod-like organelle in biological cells comprising a compound describedherein being conjugated with a drug for combined disease treatment.

In certain embodiments, a compound described herein is first dissolvedin an aqueous solution before accumulating in the rod-like organelle inbiological cells for imaging.

Also provided herein is a method for imaging rod-like organelle inbiological cells comprising accumulating a compound described herein inthe rod-like organelle in biological cells directly. In certainembodiments, the rod-like organelle in biological cells is primarycilium.

In certain embodiments, the imaging is performed using a linearfluorescence microscope under UV light excitation or a two-photonconfocal laser scanning microscope.

In certain embodiments, imaging the rod-like organelles in biologicalcells is conducted in living cells.

In certain embodiments, there is provided a method for quantitativelyanalyzing primary cilium related proteins comprising accumulating acompound described herein in the rod-like organelle in biological cellsdirectly.

In certain embodiments, there is provided a method for diagnosingprimary cilium related disease comprising using a compound describedherein as a disease diagnosis probe.

In certain embodiments, there is provided a method for targeting primarycilium in biological cells comprising using a compound described hereinas an organelle targeting specific vector or agent.

In certain embodiments, there is provided a method for treating primarycilium related disease comprising administering a compound describedherein in conjugation with a drug for combined treatment.

The presently claimed invention is further illustrated by the followingexperiments or embodiments which should be understood that the subjectmatters disclosed in the experiments or embodiments may only be used forillustrative purpose but are not intended to limit the scope of thepresently claimed invention:

Materials and Methods

Synthesis and Characterization of HGEu001 and HGEu002

General Information for the Synthesis.

Tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile (CH₃CN) andN,N-diisopropylethylamine (DIPEA) were dried over calcium hydride(CaH₂). All reactions were carried out with anhydrous solvents undernitrogen atmosphere, unless otherwise specified. All the reagents wereobtained commercially with high quality and used without furtherpurification. Reactions were monitored by thin-layer chromatography(TLC) which was carried out on silica gel plates (0.25 mm, 60F-254) byusing UV light as visualizing method. Flash column chromatography wascarried out on 200-300 mesh silica gel. ¹H and ¹³C NMR spectra wererecorded on a 400 (¹H: 400 MHz, ¹³C: 100 MHz) spectrometer. Thefollowing abbreviations were used to explain the multiplicities:s=singlet, d=doublet, t=triplet, q=quartet, dd=doublet of doublets,m=multiplet, br=broad. High resolution mass spectra were obtained froman ESI or MALDI-TOF mass spectrometer. The synthetic route for theprimary cilium specific probe HGEu001 and its motif complex. HGEu002 isshown in FIG. 29.

Synthesis of N-(4-iodophenyl)-4-(trifluoromethyl)benzamide (Compound 2)

The solution of 4-iodoaniline (5 g, 46.23 mmol) and DIPEA (13.42 mL,77.06 mmol) in DCM (200 mL), 4-(trifluoromethyl)benzoyl chloride (5.72mL, 38.53 mmol) was added dropwise at 0° C. in 30 min. The resultingsolution was stirred for 12 hours at room temperature. The solvent ofthe resulting mixture was concentrated to 100 mL, and the whiteprecipitate was collected as product after filtration. (13.41 g, 34.29mmol, yield=89%) ¹H NMR (400 MHz, DMSO-d₆): δ10.57 (s, 1H), 8.13 (d, J=4Hz, 2H), 7.92 (d, J=4 Hz, 2H), 7.72 (d, J=4 Hz, 2H), 7.63 (d, J=4 Hz,2H); (FIG. 16) ¹³C NMR (100 MHz, DMSO-d₆): δ164.5, 138.6, 138.5, 137.3,131.6, 131.3, 128.6, 125.4, 125.3, 125.2, 122.5, 87.9; (FIG. 17) HRMS(MALDI-TOF) m/z calcd. for C₁₄H₁₀F₃INO [M+H]⁺ 391.9759 found 391.9761.

Synthesis ofN-(4-((2-(hydroxymethyl)pyridin-4-yl)ethynyl)phenyl)benzamide (Compound3)

(4-ethynylpyridin-2-yl)methanol (0.92 g, 6.8 mmol) was added into thesolution of N-(4-iodophenyl)benzamide (Compound 1) (3.36 g, 10.4 mmol),Pd(PPh₃)₂Cl₂ (136 mg, 0.21 mmol), CuI (80 mg, 0.42 mmol) and DIPEA (2.0mL) in freshly distilled THF (200 mL). The resulting mixture was stirredat 45° C. for 6 hours under protection of N₂ gas. Silica gel flashcolumn chromatography (DCM:MeOH=30:1) of the concentrated residue gave awhite solid as the product. (2.16 g, 6.4 mmol, yield=94%) ¹H NMR (400MHz, DMSO-d₆): δ10.49 (s, 1H), 8.52 (d, J=2 Hz, 1H), 7.96 (d, J=4 Hz,2H), 7.90 (dd, J₁=4 Hz, J₂=8 Hz, 2H), 7.63-7.60 (m, 3H), 7.57-7.53 (m,3H), 7.38 (d, J=2 Hz, 1H), 5.52 (br, 1H), 4.58 (s, 2H); (FIG. 18) ¹³CNMR (100 MHz, DMSO-d₆): δ165.8, 162.5, 148,9, 140.4, 134.7, 132.4,131.8, 130.8, 128.4, 127.7, 123.2, 121.4, 120.1, 115.9, 93.6, 86.6,63.9, 53.5; (FIG. 19) HRMS (MALDI-TOF) m/z calcd. for C₂₁H₁₇N₂O₂ [M+H]⁺329.1290 found 329.1295.

Synthesis ofN-(4((2-(hydroxymethyl)pyridin-4-yl)ethynyl)phenyl)-4-(trifluoro methyl)benzamide (Compound 4)

(4-ethynylpyridin-2-yl)methanol (1.13 g, 8.52 mmol) was added into thesolution of N-(4-iodophenyl)-4-(trifluoromethyl)benzamide (Compound 2)(4 g, 10.22 mmol), Pd(PPh₃)₄ (197 mg, 0.17 mmol), CuI (65 mg, 0.34 mmol)and DIPEA (20 mL) in freshly distilled THF (200 mL), The resultingmixture was stirred at 45° C. for 6 hours under protection of N₂ gas.Silica gel flash column chromatography (DCM:MeOH=30:1) of theconcentrated residue gave a white solid as the product. (3.10 g, 7.84mmol, yield=92%) ¹H NMR (400 MHz, DMSO-d₆): δ10.71 (s, 1H), 8.52 (d, J=4Hz, 1H), 8.15 (d, J=4 Hz, 2H), 7.93 (d, J=4 Hz, 2H), 7.90 (d, J=4 Hz,2H), 7.64 (d, J=4 Hz, 2H), 7.55 (s, 1H), 7.36 (d, J=2 Hz, 1H), 5.54 (t,J=4 Hz, 1H), 4.14 (d, J=2 Hz, 2H); (FIG. 20) ¹³C NMR (100 MHz, DMSO-d₆):δ164.7, 162.5, 148.9, 140.0, 138.5, 132.5, 131.7, 131.3, 130.70, 128.7,125.4, 125.2, 123.2, 122.5, 121.4, 120.2, 116.3, 93.4, 86.6, 63.9; (FIG.21) HRMS (MALDI-TOF) m/z calcd. for C₂₂H₁₆F₃N₂O₂ [M+H]⁺ 397.1164 found397.1168.

Synthesis of2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tris(N-(tert-butyl)acetamide) (Compound 5)

2-promo-N-(tert-butyl)acetamide (10.1 g, 52.2 mmol) was added into thesolution of 1,4,7,10-tetraazacyclododecane (3.0 g, 17.4 mmol) inanhydrous acetonitrile (80 mL), followed by NaHCO₃ (21.9 g, 261 mmol).The resulting solution was stirred at room temperature for 24 hours.After filtration of the resulting mixture, filtrate was concentrated andrecrystallized from hot water to obtain a white solid as the product.(4.8 g, 8.7 mmol, yield=50%) ¹H NMR (400 MHz, CDCl₃): δ6.78 (s, 1H),6.68 (s, 2H), 3.05 (s, 4H), 3.05 (s, 2H), 2.70 (m, 16H), 1.37 (s, 18H)1.36 (s, 9H); (FIG. 22) ¹³C NMR (100 MHz, CDCl₃): δ170.1, 170.0, 60.5,59.6, 53.4, 52.9, 52.3, 51.1, 50.9, 46.7, 28.9, 28.8; (FIG. 23) HRMS(MALDI-TOF) m/z calcd. For C₂₆H₅₄N₇O₃ [M+H]⁺ 512.4288 found 512.4285.

Synthesis of2,2′,2″-(10-((4-((4-benzamidophenyl)ethynyl)pyridin-2-yl)methyl)-1,4,7,10-tetranzacyclododecane-1,4,7-triyl)tris(N-(tert-butyl)acetamide)(HGL001)

Methanesulfonyl chloride (0.22 mL, 2.73 mmol) was added into a stirredsolution of N-(4((2-(hydroxymethyl)pyridin-4-yl)ethynyl)phenyl)benzamide(Compound 3) (300 mg, 0.91 mmol) in anhydrous DCM (150 mL) and DIPEA(1.59 mL, 9.11 mmol). The resulting mixture was stirred at roomtemperature for 3 hours. The solution was then washed with saturatedNaHCO₃ solution, saturated NH₄Cl solution and brine. The organic layerwas dried over anhydrous Na₂SO₄ and concentrated to give a pale yellowsolid as the intermediate compound,(4-((4-benzamidophenyl)ethynyl)pyridin-2-yl)methyl methanesulfonate,which was directly used in the next step without further purification.The pale yellow solid was dissolved in dry CH₃CN (50 mL).2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tris(N-(tert-butyl)acetamide)(Compound 5, 0.50 g, 0.61 mmol) and anhydrous K₂CO₃ (1.26 g, 9.1 mmol)added. The resulting mixture was stirred at 50° C. for 12 hours under N₂gas. The solids were filtered off, and the filtrate was concentrated.Silica gel flesh column chromatography (CH₂Cl₂:MeOH=12:1) of the residuegave a pale yellow solid as the product (378 mg, 0.46 mmol, yield=75%).¹H NMR (400 MHz, DMSO-d₆): δ10.53 (s, 1H), 8.38 (d, J=2 Hz, 1H), 7.97(d, J=4 Hz, 2H), 7.94 (d, J=4 Hz, 2H), 7.83 (br, 2H), 7.60-7.53 (m, 7H),7.38 (d, J=2 Hz, 1H), 3.66 (br, 2H), 3.20-2.07 (m, 22 J), 1.30 (s, 9H),1.22 (s, 18H); (FIG. 24) ¹³C NMR (100 MHz, DMSO-d₆): δ170.5, 169.9,165.9, 158.7, 149.0, 140.5, 134.7, 132.3, 131.8, 131.1, 128.5, 128.2,127.8, 125.1, 123.5, 120.1, 115.9, 93.9, 86.3, 58.1, 57.9, 57.2, 50.4,50.3, 28.3, 28.1; (FIG. 25) HRMS (MALDI-TOF) m/z calcd. for C₄₇H₆₈N₉O₄[M+H]⁺ 822.5394, found 822.5390.

Synthesis of2,2′,2″-(10((4-((4-(4-(trifluoromethyl)benzamido)phenyl)ethynyl)pyridin-2-yl)methyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tris(N-(tert-butyl)acetamide)(HGL002)

Methanesulfonyl chloride (0.18 mL, 2.28 mmol) was added into a stirredsolution ofN-(4-((2-(hydroxymethyl)pyridin-4-yl)ethynyl)phenyl)-4-(trifluoromethyl)benzamide(Compound 4) (300 mg, 0.76 mmol) in anhydrous DCM (150 mL) and DIPEA(1.33 mL, 7.61 mmol).The resulting mixture was stirred at roomtemperature for 3 hours. After that the solution was then washed withsaturated NaHCO₃ solution, saturated NH₄Cl solution and brine. Theorganic layer was dried over anhydrous Na₂SO₄ and concentrated to give apale yellow solid as the intermediate compound,(4-((4-(4-(trifluoromethyl)benzamido)phenyl)ethynyl)pyridin-2-yl)methylmethanesulfonate, which was directly used in the next step withoutfurther purification. The pale yellow solid was dissolved in dry CH₃CN(50 mL).2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tris(N-(tert-butyl)acetamide)(Compound 5, 0.42 g, 0.51 mmol) and anhydrous K₂CO₃ (1.05 g, 7.6 mmol)were added. The resulting mixture was stirred at 50° C. for 12 hoursunder N₂ gas. The solids were filtered off, and the filtrate wasconcentrated. Silica gel flesh column chromatography (CH₂Cl₂:MeOH=12:1)of the residue gave a pale yellow solid as the product (354 mg, 0.40mmol, yield=78%). ¹H NMR (400 MHz, DMSO-d₆): δ10:78 (s, 1H), 8.38 (d,J=2 Hz, 1H), 8.18 (s, 1H), 8.16 (s, 1H), 7.91 (d, J=4 Hz, 4H), 7.82 (br,2H), 7.62 (d, J=8 Hz, 2H), 7.57 (d, J=4 Hz, 2H), 7.39 (d, J=2 Hz, 1H),3.71 (br, 2H), 2.87-2.17 (m, 22H), 1.29 (s, 9H), 1.27 (s, 18H); (FIG.26) ¹³C NMR (100 MHz, DMSO-d₆): 170.5, 169.8, 164.7, 158.6, 148.9,140.1, 138.4, 132.3, 131.6, 131.3, 131.0, 128.7, 125.4, 125.2,125.1123.4, 122.5, 120.2, 116.2, 93.7, 86.4, 58.1, 57.9, 57.2, 54.9,50.4, 50.3, 49.4, 28.3, 28.1; (FIG. 27) HRMS (MALDI-TOF) m/z calcd. forC₄₈H₆₇F₃N₉O4 [M+H]⁺ 890.5268 found 890.5264.

Synthesis of Complex HGEu001

Europium (III) chloride hexahydrate (77 mg, 0.21 mmol) was added to thesolution of the ligand (HGL001, 0.20 mmol) in MeOH/H₂O (100 mL,v:v=1:1). The resulting solution was maintained in a pH range of 6.0-6.5with NaOH solution (0.4 M) and stirred at room temperature for 24 hours.The solvents were removed under vacuum; the residue was dissolved in 1mL of methanol and dropped into ethyl ether (50 mL). The precipitateswere filtered, washed with diethyl ether and dried under vacuum at roomtemperature. White solids were collected as the products. (222 mg, 0.19mmol, yield=90%). HRMS (+ESI) m/z calcd. for C₄₀H₆₅EuN₉O₄ [M−H₂O−2H]⁺972.4372, found 972.4378, calcd. for C₄₇H₆₆ClEuN₉O₄ [M−H₂O−H+Cl⁻]⁺1008.4139, found 1008.4119, calcd. for C₄₇H₆₇Cl₂EuN₉O₄ [M−H₂O+2Cl]⁺1044.3905, found 1044.3882 (FIG. 8) HPLC characterization: Retentiontime=15.20 min. (Table 1 and FIGS. 7A-7B). The chemical structure ofHGEu001 can be represented by the following formula:

wherein X is H; Ln is Eu.

Synthesis of Complex HGEu002

HGEu002 was obtained from ligand HGL002 with same procedures as HGEu001(204 mg, 0.19 mmol, yield=95%). HRMS (+ESI) m/z m/z calcd. forC₄₈H₆₅EuF₃N₉O₄ [M−H]²⁺ m/z=1041.4324/2=520.7162, found 520.7186. (FIG.9); HPLC characterization: Retention time=15.36 min, (Table 1 and FIGS.7A-7B). The chemical structure of HGEu002 can be represented by theformula (II), wherein X is CF₃; Ln is Eu.

TABLE 1 Solvent gradient of HPLC for the characterization of HGEu001 andHGEu002. Time/min 0.05% TFA in water/% 0.05% TFA in CH₃CN/% 0.0 90 10 590 10 15 40 60 20 90 10 25 90 10 Flow rate = 1 mL/min

Synthesis of Complex of HGEu001-Coumarin

An exemplary synthesis of HGEu001-Coumarin, HGEu001-Por, HGEu001-EDECRINand HGEu001-Biotin is shown in FIGS. 37a and 37b . Referring to FIG. 37a, Compound 6 was obtained by mesylating the chromophore alcohol Compound3 and it was used in the next step without purification. Monoalkylationof2,2′-(1,4,7,10-tetraazacyclododecane-1,7-diyl)bis(N-(tert-butyl)acetamide)(Compound 7) with Compound 6 afforded Compound 8 with an overall yieldof 52% over the first two steps. Compound 8 was then reacted withCompound 9 under basic conditions to afford Compound 10 in a yield of71%. Compound 10 was then deprotected by reaction with 1 M HCl indioxane to afford the key intermediate HGL001-NH₂ in a yield of 97%.Europium (III) complexation of HGL001-NH₂ obtained the europium (III)complex HGEu001-NH₂. Finally, the lanthanide complexes HGEu001-Coumarin,HGEu001-Por, HGEu001-EDECRIN, and HGEu001-Biotin were obtained byreaction of HGEu001-NIH with the corresponding n-hydroxysuccinimide(NHS) esters: Coumarin-NHS (reaction f, FIG. 37b ), Por-NHS (reaction g,FIG. 37b ), EDECRIN-NHS (reaction h, FIG. 37b ), and Biotin-NHS(reaction i, FIG. 37b ), respectively.

HGEu001-Coumarin, HGEu001-Por, HGEu001-EDECRIN, and HGEu001-Biotin wereeach characterized by high resolution mass spectroscopy (HRMS). Theresults of these tests are shown below and confirm that the desiredproducts were synthesized.

HGEu001-NH₂. Yield=96%; HRMS (+ESI) m/z calcd. for C₄₉H₇₀EuN₁₀O₄[M−H₂O−2H]⁺ 1015.4794, found 1015.4783.

HGEu001-Coumarin. Yield=87%; HRMS (+ESI) m/z calcd. for C₆₃H₈₄EuN₁₁O₇[M−H₂O−H]⁺ 12.59.5767/2=629.7883, found 629.7886.

HGEu001-Por. Yield=90%; HRMS (+ESI) m/z calcd. for C₁₀₃H₁₁₆EuN₁₄O₁₄[M−H₂O−H]²⁺ 1925.8008/2=962.9004, found 962.9009.

HGEu001-EDECRIN. Yield=85%; HRMS (+ESI) m/z calcd. for HGEu001-Biotin.Yield=92%; HRMS (+ESI) m/z calcd. for C₅₉H₈₅EuN₁₂O₆S [M−H₂O−H]²⁺1242.5648/2=621.2824, found 621.2819.

Photophysical Studies

UV-Visible absorption spectra in the spectral range 200 to 1100 nm wererecorded by an HP Agilent UV-8453 Spectrophotometer. The emissionspectra and the emission decay lifetimes of HGEu001 and HGEu002 weremeasured by Horiba Fluorolog-3 spectrophotometer and also measured byEdinburgh instrument ELS920 spectrophotometer for cross checking. (Twospectrophotometers are equipped with a 450 W continuous xenon lamp forsteady state emission measurement, 60 W xenon flashlamp for emissionlife time measurement and an UV-Vis PMT detector—Hamamatsu—R₉₂₈ cooled216 at −20° C.)

Stability Test Via Europium Emission Titration

Kinetic stability of the europium complexes were conducted toinvestigate the stability of the complexes in the diluted solution andin the present of 100 times of [EDTA]²⁻ and Ca²⁺. EDTA was chose as acompetitive ligand for the Eu³⁺ ion. Ca²⁺ cation serves as competitiveions for the ligand which can bind to the cyclen based macrocyclicligand. 10 μM of europium complex was co-incubated 1 mM of EDTA or Ca²⁺at room temperature, and the europium emission spectra were measured atdifferent time point (1, 24 and 48 hours).

Materials

Tissue Culture

Human cervical cancer HeLa cells were grown in Dulbecco's Modified EagleMedium (DMEM). Human lung normal diploid fibroblasts MRC-5 andneuroblastoma cells SK-N-SH were provided by Cell resource center ofShanghai Institute of Biological Sciences, Chinese Academy of Sciences;and cultured in MEM (GIBCO 41500034); Human derived liver cells QSG-7701cells were grown in RPMI-1640 (GIBCO 31800022); all cells weresupplemented with 10% (v/v) fetal bovine serum, 1% penicillin andstreptomycin at 37° C. and 5% CO₂.

MTT Cell Cytotoxicity Assays

HeLa, SKA-SH, QSG-7701 or MRC-5 cells treated with testing complexes for24 hours were further incubated with MTT, 3-(4,5-dimethylthiazol-2-yl)-2and 5-diphenyltetrazolium bromide (0.5 mg; ml) for 4 hours, to produceformazan during cell metabolism. Then, formazan was thoroughly dissolvedby dimethyl sulfoxide (DMSO), and the absorbance of solutions wasmeasured in Bio-Rad iMark microplate reader (570 nm). Quadruplicateswere performed. Data analysis and plotting were operated by the GraphPadPrism 5 software.

Two-Photon Confocal in Vitro Imaging

Cells were seeded on coverslip in 35-mm culture dishes overnight. Andthen incubated with HGEu001 or HGEu002 (10 μM) for 6 hours, and thecells were washed by PBS 3 times before imaging. For the two-photon timelapses in vitro images of HGEu001 and HGEu002 were observed withdifferent incubation time (3, 6, 18 and 24 hours). Then the unabsorbedcomplexes were washed out with PBS buffer and the cells were subject tomicroscopic imaging. The in vitro imaging of HGEu001 and HGEu002 wereundertaken on a linear fluorescence microscopy under 375 nm UV lightexcitation or a confocal laser scanning microscope, Leica TCS SP8,equipped with a Ti:sapphire laser (Libra II, coherent). The excitationbeam produced by 690 nm to 1080 nm (fs laser) was focused on theadherent cells through a 63× oil immersion objective.

3D images obtained by the Z-stacks form the two-photon confocalmicroscopy and the 3D reconstruction was done with built-in programs ofLeica TCS SP8 confocal microscope.

Co-Localization Imaging

(a) 3D Co-Localization Imaging of HGEu001 and HGEu002 with Primary CiliaMarkers ARTL13B and IFT88.

Full length ARTL13B (ADP-ribosylation factor-like protein 13B) was PCRamplified from cDNA library and inserted into multiple cloning sites(MCS) of pEGFP-C3 (CLONETECH, #6082-1) using restriction digestion sitesof XhoI/EcorI for GFP-ARTL13B expression. The positive recombinant wasselected through kanamycin proved by sequencing. PlasmidmEmerald-IFT88-N-18 (intraflagellar transport protein 88 homolog)expressing GFP-IFT88 was a gift from Michael Davidson (Addgene plasmid#54125). Plasmids were amplified in E. coli (DH5α) and purified using aStarPrep Plasmid Miniprep Kit (Genstar). Both GFP-ARL13B and GFP-ITF88for primary cilium tracking were expressed in HeLa cells throughlipofectamine2000 (Cat. No.11668-019, Invitrogen) mediated transfection.Briefly, a 3.5 cm dish of HeLa cells (with 70-80% confluent) wastransfected with 4 μg DNA plus 4 μL lipotectamine2000 mixture accordingto the manufacturer instructions. After incubation for 8 hours, 10 μM ofHGEu001 or HGEu002 were added and incubated for 6 hours.

(b) Co-Localization Imaging of HGEu001 and HGEu002 with Organelles.

Live cell labeling probes of organelles (mitochondria, lysosome andGolgi apparatus) MitoTracker® Green FM (M-7514), LysoTracker® GreenDND-26 (L-7526) and GolgiTracker® Oregon Green (W6748, Wheat GermAgglutinin) were respectively purchased from ThermoFisher Scientic Inc.and stocked in −20° C.

Three dishes of HeLa cells (1×10⁵) were first incubated with 10 μMHGEu001/HGEu002 for 6 hours. Then organelles probes (50 nM, each) wereadded in parallel to the cells and further incubated for 15 minutes. Thecells were washed by PBS 3 times before imaging on the linearfluorescence microscopy. Under the excitation of UV light (375 nm)emission from the channel (610-630 nm) were collected for the emissionsignals from HGEu001/HGEu002. Under 488 nm blue light excitation,imaging channel in the range between 505 and 555 nm were collected fromthe emission signal of organelles dyes.

Absorption and Emission Spectra of HGEu001-Coumarin and HGEu001-Por

UV-Visible absorption spectra of HGEu001-Coumarin and HGEu001-Por wererecorded by an HP Agilent UV-8453 Spectrophotometer. The emissionspectra of HGEu001-Coumarin and HGEu001-Por were measured by HoribaFluorolog-3 spectrophotometer, which was equipped with a 450 Wcontinuous xenon lamp and a UV-Vis PMT detector—Hamamatsu—R928 cooled216 at −20° C. for steady state emission measurement.

Protein Enrichment Using HGEu001-Biotin and HGEu001-EDECRIN.

a) Protein Labeling Using HGEu001-EDECRIN

HGEu001-EDECRIN was reacted with cell lysate at 4° C./16° C./37° C. for10 min. The protein mixture was then treated with SDS-PAGE loadingbuffer. The resulting mixture were boiled for 10 min and centrifuged at14000 rpm for 5 minutes. Then the protein mixture was loaded onto onewell of a gel and separated by SDS-PAGE using a Hoefer SE400 VerticalElectrophoresis System (18×16 cm). The gel was stained with Coomassiebrilliant blue 6250 and imaged. Florescence images of the gel were takenunder UV light excitation. The fluorescent protein band was excised andthen destained in 50% (v/v) acetonitrile, dehydrated with gradientacetonitrile (50-70%) and then cysteine-alkylated bydithiothreitol/iodoacetamide. MS compatible peptides of each of thebands were generated by In-Gel trypsin digestion (Trypsin Gold, V5280,Mass Spectrometry Grade 100 mg (1 vial), Promega) at 37° C. for 16 hoursand extracted using 70% acetonitrile (with 0.02% trifluoroacetic acid)three times. The extracts were combined and vacuum dried, resuspended inLC buffer (95% H2O, 5% acetonitrile, 0.1% formic acid) and readied formass spectrometry analysis.

b) Protein Pull-Down Using HGEu001-Biotin

HGEu001-Biotin was incubated with cell lysate at 37° C. for 10 min,HGEu001-Biotin binding protein was enriched using Monomeric AvidinUltraLink resin using home-made micro-columns. The proteins were furtheranalyzed by SDS-Gel analysis. Since the amount of enriched protein wastoo small, there was no one clear band could be seen by coomassie bluestaining method. The protein band, which had the same position withfluorescent band of HGEu001-EDECREN labeling and imaging assay asdescribe above, was excised and further treated for nLC-MS/MS analysis.

Results

Two europium complexes HGEu001 and HGEu002 were synthesized by similarprocedures as shown in Scheme 1 (FIG. 29). Recrystallization of thecrude europium complexes with diethyl ether gave HGEu001 and HGEu002 ina yield of ˜95%. All the intermediates were well characterized by¹H-NMR, ¹³C-NMR and HRMS. The europium complexes, HGEu001 and HGEu002,were purified and verified by high-performance liquid chromatography andHRMS (FIGS. 7A-7B, 8-9). The strong absorption bands of the complexHGEu001 in water can be found peaking at 340 nm (ε>20,000 cm⁻¹, Table2), corresponding to the π→π* transition (FIG. 10).

The photo-physical properties of HGEu001 and HGEu002 were recorded inaqueous solution and have similar europium emission quantum yields(ϕ=˜10%). The five europium f-f emission ⁵D₀→⁷F_(J) bands (J=0-4) wereobserved and the ratio of these f-f transition intensity is consistentwith literature reports of a typical cyclen-based europium complexeswith a D2h symmetry (FIGS. 1A-1B, FIGS. 11A-11B and Table 2). Showingsimilar molecular structures and photo-physical properties, the toxicityof HGEu001 and HGEu002 is also similarly low in HeLa, SK-N-SH, QSG-7701and MRC-5 cells (IC50 are ˜390-440 μM, Table 3). However, the in vitrosubcellular localization of HGEu001 and HGEu002 is vastly different,especially HGEu001—the red emission of HGEu001 in vitro did notcorrelate with common commercial organelle-specific markers, such asmitochondria, lysosome and Golgi apparatus in fluorescence microscopy(FIGS. 2A-2I).

TABLE 2 Photophysical properties of the europium complexes HGEu001 andHGEu002. λ_(max)/ ε/M⁻¹ τ(H₂O)/ τ(D₂O)/ Φ_(L) ^(Eu)/ Complex nm ^([a])cm⁻¹ ^([a]) ms ^([b]) ms ^([b]) q^([c]) % ^([d]) HGEu001 327 23400 0.561.78 0.9 10.5 HGEu002 327 22200 0.56 1.78 0.9 10.3 ^([a]) Absorptioncoefficient in H₂O, 298K; ^([b]) Europium emission decay in FIGS.12A-12B (λ_(em) = 614 nm. ⁵D₀→⁷F₂. λ_(ex) = 340 nm); ^([c])Derivedhydration numbers, q (±20%) q = 1.2[(k(H₂O) k(D₂O)) − (0.25 + 0.07x)] (k= τ⁻¹, x = number of carbonyl-bound amide NH oscillators); ^([d])Overall europium emission quantum yield in H₂O, by-integrated sphere.

TABLE 3 Cytotoxicity of the complexes HGEu001 and HGEu002 against HeLa,SK-N-SH, QSG-7701 find MRC-5 cell line. (IC₅₀/μM) Complex HeLa SK-N-SHQSG-7701 MRC-5 HGEu001 411 389 395 395 HGEu002 417 437 410 402Incubation time = 24 hours; Raw data are shown in FIGS. 13A-13B.

DISCUSSION

To further confirm the reputation of the primary cilia-specific in vitrosubcellular localization of HGEu001, in vitro imaging of HGEu001 wasperformed on a multi-photon confocal microscope (FIGS. 3A-3H). With 3hours of incubation in HeLa cells, red europium emission of HGEu001 wasfound in a particularly focused area in the primary cilium under twophoton excitation (λ_(ex)=700 nm, FIGS. 3A-3H) and optimum emissionintensity lasted for 24 hours. However, the red emission of HGEu002 wasfound dispersed in the cytoplasm (FIGS. 14A-14H and 15A-15F). The invitro subcellular localization of HGEu001 and HGEu002 are the same innumber of cell lines, such as HeLa, SK-N-SH and MRC-5 (FIGS. 13A-13B).

Aiming to confirm this subcellular localization of HGEu001, a positivecontrol has been done. The co-localization experiments have been done byGreen Fluorescent Protein-fused with primary cilia marker ARL13B(GFP-ARL13B). The advantages of using Green Fluorescent Protein “GFP”are its bright luminescence and availability to trace the localizationof cellular proteins, such as antibody, at the targets in vitro.However, GFP also gives scientists a lot of hassle in molecular imaging,such as its complicated process in cloning, transfection and their broademission profile and short emission lifetime presents a low signal tonoise ratio problem. There are no commercially available simple primarycilia markers, therefore, the present invention could only compare thesubcellular localization between HGEu001 and GFP-ARL13B (ARL13B is thecilium-specific protein required for culinary axoneme structure) andwith HGEu002 as a negative control (FIGS. 2A-2I).

The perfect overlapping of the in vitro red emission from HGEu001(dosage concentration=10 μM, incubated for 6 hour after GFP wastransfected and expressed) with the green emission from GFP-ARL13B(transfection procedure see ESI) is shown in FIGS. 4A-4H. The yellowemission of the merged images is shown as well (HGEu001, λ_(ex) 700 nm,BP 550-665 nm, and GFP-ARL13B λ_(ex)=488 nm, BP 505-555 nm). On theother hand, the red emission of HGEu002 and green emission of GFP-ARL13Bare localized in different parts in the cytoplasm and only slight yellowemission can be found in their merged images (FIG. 4G). This can confirmthe selectivity of HGEu001 in primary cilia, but not HGEu002. As primarycilium is a rod-like organelle, two dimensional florescence images orconfocal images are not good enough to show the specificity of theHGEu001 localization. With their two photon induced emission properties,three dimensional confocal images of HGEu001 and HGEu002 were obtainedin HeLa cells by two photon confocal microscope (z stack) withexcitation at 700 nm (FIGS. 5A-5H and 6A-6D). Red emission can be foundin the HeLa cells and is shown as the red column (FIG. 5A). In controlexperiments, HGEu002 did not show any europium emission in the rod-likeorganelles (FIG. 5B). However, HGEu002 appears to localize in certainpart of the cytoplasm instead of primary cilia.

To further confirm the selective primary cilia subcellular localizationof HGEu001, three dimensional red in vitro imaging of HGEu001 and itsmotif control HGEu002 were compared with the primary cilia-specificgreen GFP-ARL13B and also with another primary cilia marker GFP-IFT88(IFT88 is the component of IFT complex B involved in cilium biogenesis)(FIGS. 6A-6D). Only HGEu001 showed the yellow merged in vitro emissionwith the co-staining with GFP-ARL13B or GFP-139IFT88. Besides, HGEu001did not show merged yellow in vitro emission with green mito-tracker.HGEu002 also did not show merged yellow in vitro emission with neitherGFP-ARL13B, GFP-IFT88 nor green mito-tracker. The 3D video of in vitroimaging in FIGS. 5A-5F and 6A-6D are shown in the supportinginformation.

In general, with comprehensive co-staining subcellular localization invitro imaging studies, the selectivity of HGEu001 in primary cilia invitro can be confirmed with good confidence. HGEu001 and HGEu002 havevery similar structures, however, only HGEu001 preferentially localizesin primary cilium. Since HGEu001 did not show any non-specific stainingon other organelles such as lysosome, mitochondria, or Golgi apparatus(FIGS. 2A-2I), binding to the structural components of primary cilium(e.g. cilium membrane, microtubules and associated proteins) or itsassociated factors, shall be a possible explanation for the specificallylocalization on the primary cilium of HGEu001.

The binding mechanism of the present molecules could be studied byanalytical means such as proteomics mass spectrometry. Hence the presentinvention provides a blueprint or strategy for developing a set ofprimary cilia targeting probes with special functions or deliveringpurposes towards primary cilia by simple conjugation with the HGEu001motif structure. In fact, the imaging protocol established in thepresent invention can also be used to validate the specific binding ofany future probes to primary cilium, with emphasis on visualizing therod-like structure by 3D imaging.

There is provided in the present invention a direct imaging tool forprimary cilia which is specific, and can be excited by light in the nearinfrared (NIR) region. Comprehensive in vitro studies and severalcontrol experiments were done to confirm the specific primary ciliumlocalization of HGEu001, showing a great degree of agreement withGFP-conjugated primary cilia markers ARL13B and IFT88 in co-localizationexperiments. These novel primary cilium marker could help to understandthe functions and roles of primary cilium in life science, such astumorigenesis.

There is also provided in the present invention a possible template fordesigning primary cilium-specific molecules with potential modificationto become target-specific drug delivery vehicles to help understand thefunctions of primary cilium and evolve into cancer or diseases treatmentapplications. A molecular library, HGEu003-HGEu016 (structures shown inFIG. 28) based on the complex HGEu001 has been developed and primarycilium imaging screening has been undertaken. Proteomic massspectroscopy studies could also be carried out to evaluate the specificbinding of HGEu001 with particular proteins in primary cilium.

Primary Cilium Imaging Marker with Expanded Excitation and EmissionWavelength.

The excitation of the primary cilium imaging marker HGEu001 can beaccomplished by UV light or NIR light (e.g., using a femtosecond laser).Consequently, its application may be limited by the configuration and/oravailability of the imaging microscopy instrument(s) on hand. Moreover,the fluorescent filter of some fluorescence microscopy instruments isnot turntable, which means that if UV excitation is applied, redemission cannot be collected due to the configuration of the microscopyinstrument. For NIR femtosecond laser excitation, the cost offemtosecond laser instruments is still prohibitively high, which haslimited the availability of these instruments in research laborites. Toextend the range of microscopy instruments that the primary ciliumimaging markers described herein can be used in connection with and thustheir accessibility, additional excitation and emission wavelengths canbe achieved using certain embodiments of the compound of Formula (IV)(e.g., HGEu001-Coumarin and HGEu001-Por). Using such cilium imagingmarkers removes the need to change the configuration of commonly usedand widely available fluorescent/confocal microscopy instruments. Theabsorption and emission spectra of HGEu001-Coumarin and HGEu001-Por wereexamined in aqueous solution. (FIGS. 31a,b, and c and FIGS. 32a,b, and c) HGEu001-Coumarin shows an additional absorption band located at 430 nmcompared to HGEu001, which belongs to the π→π* of coumarin moiety. Inthe emission spectra, with 330 nm excitation, both characteristicemission bands from europium (III) ion and coumarin moiety can beobserved. With 430 nm excitation, only the coumarin emission band can beobserved since light having a wavelength of 430 nm cannot exciteHGEu001. Similar results can be observed from HGEu001-Por.HGEu001-Coumain and HGEu001-Por provide new excitation and emissionchannels for optical imaging, which can expand the application of thechemically synthesized primary cilium imaging markers described hereinto all type of microscopy.

Primary Cilium Related Protein Enrichment and Analysis

Two new exemplary probes HGEu001-EDECRIN and HGEu001-Biotin weresynthesized for primary cilium related protein enrichment. Thehypothesis was that HGEu001 allows for specific imaging of primary ciliadue to the specific binding of HGEu001 with primary cilium relatedproteins. From SIDS-PAGE gel analysis of HGEu001-EDECRIN labeling assay,a number of protein bands can be seen from the image of the gel stainedby Coomassie blue since the cell lysate contains all the proteins of thecells. However, in the fluorescent imaging using UV excitation only oneemission band can be clearly observed. Fluorescent imaging ofHGEu001-Biotin was not performed, because the interaction betweenHGEu001-Biotin and its binding protein target is noncovalent binding,and the HGEu001-Biotin-protein binding partner were destroyed by theSDS-PAGE treating conditions. (FIG. 34) The protein detection limitationof Coomassie blue staining method is too pool that there is no clearband can be seen of gel of HGEu001-Biotin enriched protein. However, theLC-MS/MS analysis identified 93 and 391 proteins by HGEu001-Biotin andHGEu001-EDECRIN respectively. Comparing the proteins identified, therewere 72 proteins that overlapped using the two different methods. (FIG.35) Primary cilium related tubulin and actin proteins could be found inthese 72 overlapping proteins. In addition, one HGEu001-EDECRIN labeledpeptide was found by the primary data analysis. The peptide sequenceidentified belonged to amino acids 71-103 of human beta-actin protein(GenBank: ABD66582.1), which is a known primary cilium related protein.

INDUSTRIAL APPLICABILITY

The presently claimed invention provides a water-soluble, simple, stabletris(N-(tert-butyl)acetamide) cyclen-based europium complex HGEu001which exhibits the specific subcellular localization in the primarycilium with a quantum yield as high as 10% in water and a lifetime of0.56 ms lifetime. In particular, the present invention providessimplicity of the design and synthesis of a complex HGEu001. The presentmolecules or complexes are useful in imaging primary cilium inbiological cells which serve as an organelle-specific probe for primarycilium in order to identify any disorder, disease or cancer that isassociated with this particular organelle. Currently there is no suchorganelle-specific probe in place, which the present invention can meetthis need.

What is claimed is:
 1. A compound represented by formula (IV):

wherein m is an integer selected from 0, 1, 2 or 3; X is apharmaceutically acceptable anion; Ln is Eu, Tb, Gd, Yb, Er, Dy, Sm, La,Ce, Pr, Nd, Pm, Tm or Y; A₁ is C, N, or Si; R₁, R₂ and R₃ areindependently selected from the group consisting of —N(R₁₂)₂;

R₄, R₅, R₆, R₇, R₈, R₉ and R₁₀ are independently selected from the groupconsisting of H, alkyl, —CF₃, —OR₁₂, and —N(R₁₂)₂; R₁₁ is hydrogen,alkyl, aryl ether, —CO₂R₁₃; or NH(C═O)R₁₄; R₁₂ and R₁₃ for each instanceis independently selected from the group consisting of hydrogen andalkyl; R₁₄ is alkyl, cycloalkyl, aryl, or heteroaryl; and n is a wholenumber selected from 1-8, wherein one of R₁, R₂ and R₃ is not N(R₁₂)₂.2. The compound of claim 1, wherein R₁ and R₃ are independently—N(H)(alkyl).
 3. The compound of claim 2, wherein A₁ is C.
 4. Thecompound of claim 2, wherein n is 4, 5, or
 6. 5. The compound of claim4, wherein R₁₁ is —NH(C═O)Ar.
 6. The compound of claim 1, wherein thecompound is represented by the formula (V):

wherein m is an integer selected from 0, 1, 2 or 3; X is apharmaceutically acceptable anion; Ln is Eu, Tb, Gd, Yb, Er, Dy, Sm, La,Ce, Pr, Nd, Pm, Tm or Y; R₂ is selected from the group consisting of:

R₁₂ for each instance is independently alkyl; and n is a whole numberselected from 1-8.
 7. The compound of claim 6, wherein each instance ofT₁₂ is tert-butyl.
 8. The compound of claim 7, wherein n is 4, 5, or 6.9. The compound of claim 1, wherein the compound is selected from thegroup consisting of:

wherein Ln is Eu, Tb, Gd, Yb, Er, Dy, Sm, La, Ce, Pr, Nd, Pm, Tm or Y;and X is a pharmaceutically acceptable anion.
 10. The compound of claim1, wherein the molecule selectively binds to primary cilium.
 11. Amethod for imaging primary cilium in a biological cell comprising thesteps of contacting the biological cell with the compound of claim 1 andimaging the biological cell.
 12. The method of claim 11, wherein theimaging is performed using linear fluorescence microscopy using UV lightexcitation or a two-photon confocal laser scanning microscope.
 13. Themethod of claim 11, wherein the imaging is done in living biologicalcells.
 14. A method for preparing a compound of claim 9, comprising thestep of: a) contacting a compound of formula (VI)

with a compound selected from the group consisting of

thereby forming the compound of claim 9.